A variety of Clostridium sp. strains that secrete neurotoxic toxins have been discovered since 1980's, and the characterization of toxins that are secreted from these strains has been made for the past 70 years.
Neurotoxic toxins derived from the Clostridium sp. strains, that is, botulinum toxins, are classified into eight types (types A to H) depending on their serological properties. Each of the toxins has a toxin protein having a size of about 150 kDa and naturally contains a complex of several non-toxic proteins bound thereto. A medium complex (300 kDa) is composed of a toxin protein and a non-toxic non-hemagglutinin protein, and a large complex (450 kDa) and a very large complex (900 kDa) are composed of the medium-sized complex bound to hemagglutinin (Sugiyama, H., Microbiol. Rev., 44:419, 1980). Such non-toxic proteins are known to function to protect the toxin from low pH and various proteases in the intestines.
The toxin is synthesized as a single polypeptide having a molecular weight of about 150 kDa in cells, and then cleaved at a position of ⅓ starting from the N-terminal end by the action of intracellular protease or treatment with an artificial enzyme such as trypsin into two units: a light chain (L; molecular weight: 50 kDa) and a heavy chain (H; molecular weight: 100 kDa). The cleaved toxin has greatly increased toxicity compared to the single polypeptide. The two units are linked to each other by a disulfide bond and have different functions. The heavy chain binds to a receptor of a target cell (Park. M. K. et al., FEMS Microbiol. Lett., 72:243, 1990) and functions to interact with a biomembrane at low pH (pH 4) to form a channel (Mantecucco, C. et al., TIBS., 18:324, 1993), and the light chain has pharmacological activity, and thus imparts permeability to cells using a detergent or interferes with the secretion of a neurotransmitter when introduced into cells by, for example, electroporation (Poulain, B. et al., Proc. Natl. Acad. Sci. USA., 85:4090, 1988).
The toxin inhibits the exocytosis of acetylcholine at the cholinergic presynapse of a neuromuscular junction to cause asthenia. It has been considered that even treatment with a very small amount of the toxin exhibits toxicity, suggesting that the toxin has any enzymatic activity (Simpson, L. L. et al., Ann. Rev. Pharmaeol. Toxicol., 26:427, 1986).
According to a recent report, the toxin has metallopeptidase activity, and its substrates include composed of synaptobrevin, syntaxin, a synaptosomal associated protein of 25 KDa (SNAP25), etc., which are the unit proteins of an exocytosis machinery complex. Each type of toxin uses one of the above-described three proteins as its substrate, and it is known that type B, D, F and G toxins cleave synaptobrevin at a specific site, type A and E toxins cleave SNAP25 at a specific site, and type C cleaves syntaxin at a specific site (Binz, T. et al., J. Biol. Chem., 265:9153, 1994).
Particularly, botulinum toxin type A is known to be soluble in a dilute aqueous solution at a pH of 4.0-6.8. It is known that a stable non-toxic protein is separated from neurotoxin at a pH of about 7 or higher, and as a result, the toxicity is gradually lost. Particularly, it is known that the toxicity decreases as pH and temperature increase.
The botulinum toxin is fatal to the human body even in small amounts and is easy to produce in large amounts. Thus, it constitutes four major bio-terror weapons together with Bacillus anthracis, Yersinia pestis and smallpox virus. However, it was found that, when botulinum toxin type A is injected at a dose that does not systematically affect the human body, it can paralyze local muscle in the injected site. Based on this characteristic, botulinum toxin type A can be used in a wide range of applications, including winkle removing agents, agents for treating spastic hemiplegia and cerebral palsy, etc. Thus, the demand for botulinum toxin type A has increased, and studies on methods of producing botulinum toxin so as to satisfy the demand have been actively conducted.
A current typical commercial product is BOTOX® (a purified neurotoxin complex of botulinum toxin type A) that is commercially available from Allergan, Inc., USA. A 100-unit vial of BOTOX® is composed of about 5 ng of a purified neurotoxin complex of botulinum toxin type A, 0.5 mg of human serum albumin and 0.9 mg of sodium chloride and is reconstituted using sterile saline without a preservative (injection of 0.9% sodium chloride). Other commercial products include Dysport® (a complex of Clostridium botulinum toxin type A and hemagglutinin, which has lactose and human serum albumin in a pharmaceutical composition containing botulinum toxin and is reconstituted using 0.9% sodium chloride before use) that is commercially available from Ipsen Ltd., UK and MyoBloc® (an injectable solution (a pH of about 5.6) comprising botulinum toxin type B, human serum albumin, sodium succinate and sodium chloride) that is commercially available from Solstice Neurosciences, Inc.
Conventional methods used to produce botulinum toxins include an acid precipitation method, a method of precipitation with salt, and a chromatographic method.
For example, Japanese Unexamined Patent Application Publication No. 1994-192296 discloses a method of producing a crystalline botulinum toxin type A by culturing Clostridium botulinum bacteria, followed by acid precipitation, extraction, addition of nuclease, and crystallization. Further, U.S. Pat. No. 5,696,077 discloses a method of producing a crystalline botulinum toxin type B by culturing Clostridium botulinum bacteria, followed by acid precipitation, extraction, ion exchange chromatography, gel filtration chromatography and crystallization.
Simpson et al. produced a botulinum toxin type A by purifying botulinum neurotoxin by gravity flow chromatography, followed by HPLC, capture using affinity resin, size exclusion chromatography, and ion (anion and cation) exchange chromatography including the use of two different ion exchange columns (Method in Enzymology, 165:76, 1988), and Wang et al. used precipitation and ion chromatography to purify a botulinum toxin type A (Dermatol Las Faci Cosm Surg., 2002:58, 2002).
Moreover, U.S. Pat. No. 6,818,409 discloses the use of ion exchange and lactose columns to purify a botulinum toxin, and U.S. Pat. No. 7,452,697 discloses a method of preparing a botulinum toxin type A by ion exchange chromatography and hydrophobic chromatography. Korean Patent Unexamined Patent Application Publication No. 2009-0091501 discloses a method of purifying a botulinum toxin by acid precipitation and anion exchange chromatography, and U.S. Patent Publication No. 2013-0156756 discloses a method of purifying a botulinum toxin by anion exchange chromatography and cation exchange chromatography.
However, the conventional methods have problems in that the use of anion exchange chromatography adversely affects the gel banding pattern of botulinum toxins (U.S. Pat. No. 7,452,697) and in that these conventional methods are difficult to apply commercially, due to a long purification time. In addition, because Clostridium botulinum that is a botulinum toxin-producing strain is an anaerobic bacterium, there is a problem in that fermentation of the bacterium should be performed in an anaerobic system, and thus it is difficult to produce botulinum toxins in large amounts. In addition, there is a problem in that the active ingredient botulinum toxin purified by the above-described purification method is not clearly separated and identified, and thus contains impurities. Additionally, the conventional methods for producing botulinum toxins have a problem in that a filtration or dialysis process is necessarily performed to purify a high-purity botulinum toxin, suggesting that the purification process is complex and difficult.
In addition, in conventional processes for producing botulinum toxin, enzymes such as DNase or RNase were used to remove nucleic acids such as DNA or RNA (see, e.g., Korean Patent No. 10-1339349, and a conventional method for producing botulinum toxin as shown in FIG. 1). However, because enzymes such as DNase or RNase are of animal origin, these enzymes have the potential to contain various disease-causing substances, particularly abnormal prion proteins of animal origin known to cause transmissible spongiform encephalopathy, and thus have problems in terms of safety.
Transmissible spongiform encephalopathy (TSE) is known as a neurodegenerative disorder causing serious degeneration of neurons, and examples thereof includes bovine spongiform encephalopathy (BSE), Scrapie, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, Kuru, transmissible mink encephalopathy, chronic wasting disease, feline spongiform encephalopathy, etc., which affect humans and animals. It was reported that BSE crosses the species barrier and affects even humans.
The agent that causes transmissible spongiform encephalopathy (TSE) has characteristics in that it has no immunogenicity and the incubation period is long. From histopathological analysis of BSE-affected bovine brain tissue, it can be seen that special spongiform vacuoles were formed in the brain due to damage to neurons and deposition of abnormal protein fibers.
The cause of TSE is an infectious protein known as the abnormal prion. Unlike general viruses that require nucleic acid, the abnormal prion is an infectious particle composed of protein alone without containing nucleic acid. Regarding TSE, it is known that, when an abnormal prion (PrPsc) that is an infectious particle binds to a normal prion (PrPc), it is converted to a pathogenic prion which is then accumulated in the brain (Prusiner SB, Alzheimer Dis Assoc Disord., 3:52-78, 1989).
Creutzfeldt-Jakob disease is a rare neurodegenerative disorder of human transmissible spongiform encephalopathy
(TSE) where the transmissible agent is apparently an abnormal isoform of a prion protein. An individual with Creutzfeldt-Jacob disease can deteriorate from apparent perfect health to akinetic mutism within six months. Thus, a potential risk may exist of acquiring a prion mediated disease, such as Creutzfeldt-Jacob disease, from the administration of a pharmaceutical composition which contains a biologic, such as a botulinum toxin, obtained using animal-derived products. Thus, if a pharmaceutical composition is prepared using drug substance produced using animal-derived components, it can subject the patient to a potential risk of receiving various pathogens or infectious agents.
Thus, a method of producing botulinum toxin by an animal product-free (APF) process is urgently needed to solve safety problems such as transmissible spongiform encephalopathy infection caused by such animal-derived components worldwide.
Under such a technical background, the present inventors have made extensive efforts to develop a method capable of preventing the risk of exposure to prion-mediated disease (transmissible spongiform encephalopathy (TSE)) and increasing the purity of botulinum toxin, and as a result, have found that when a culture of a botulinum toxin-producing strain is treated with acid to form a botulinum toxin precipitate and the formed botulinum toxin precipitate is clarified using at least one technique selected from the group consisting of depth filtration (DF), microfiltration (MF), ultrafiltration (UF), sterile filtration, membrane chromatography (MC) and centrifugation, which are pretreatment processes, followed by performing a process selected from among UF diafiltration, ammonium sulfate precipitation and hydrochloric acid precipitation, followed by purification using anion/cation-exchange chromatography, an enzymatic treatment step that uses animal products can be omitted to eliminate the risk of causing prion-mediated disease, and the purity of the botulinum toxin can be increased, thereby completing the present invention.